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1 Targeting the WASF3-CYFIP1 Complex Using Stapled Peptides

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1 Targeting the WASF3-CYFIP1 Complex Using Stapled Peptides Author Manuscript Published OnlineFirst on December 16, 2015; DOI: 10.1158/0008-5472.CAN-15-1680 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Targeting the WASF3-CYFIP1 complex using stapled peptides suppresses cancer cell invasion Yong Teng1*, Abdulaziz Bahassan1, Dayong Dong1,3, Laura E Hanold2, Xiaoou Ren1, Eileen J Kennedy2 and John K Cowell1*. 1Cancer Center, Georgia Regents University, Augusta, GA30912, USA 2 Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, University of Georgia, Athens, GA 30602. 3 Present Address; Laboratory of Vaccine and Antibody Engineering, Beijing Institute of Biotechnology, Beijing, China 100071 Running Title: Role of WASF3-CYFIP interaction in invasion Key Words: CYFIP1, WASF3, stapled peptide, cancer invasion, metastasis, therapeutics Financial support: This work was supported in part by grants from the National Institutes of Health, CA120510 (JKC) and 1K22CA154600 (EJK). * Co-corresponding Authors: Yong Teng, Tel: 7067215257, Fax: 7067211671, E-mail: [email protected]; John Cowell, Tel: 7067214381, Fax: 7067211671, E-mail: [email protected] Disclosure of Potential Conflicts of Interest The authors declare no competing financial interests. Word count = 4998 Figures = 6 Tables = 0 ABSTRACT Activation of the WASF3 protein by extracellular stimuli promotes actin cytoskeleton reorganization and facilitates cancer cell invasion, whereas WASF3 depletion suppresses invasion and metastasis. In quiescent cells, the interaction between WASF3 and a complex of proteins including CYFIP1 acts as a conformational restraint to prevent WASF3 activation. Therefore, we took advantage of this endogenous regulatory mechanism 1 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2015 American Association for Cancer Research. Author Manuscript Published OnlineFirst on December 16, 2015; DOI: 10.1158/0008-5472.CAN-15-1680 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. to investigate potential sites that disrupt WASF3 function. Here, we show that genetic knockdown of CYFIP1 in cancer cells led to the destabilization of the WASF3 complex, loss of WASF3 function, and suppressed invasion. Based on existing crystallographic data, we developed stapled peptides, referred to as WASF Helix Mimics (WAHM), that target an α-helical interface between WASF3 and CYFIP1. Treatment of highly invasive breast and prostate cancer cells with WAHM inhibitor peptides significantly reduced motility and invasion in vitro. Mechanistic investigations revealed that these inhibitors suppressed the interaction between Rac and the WASF3 complex, which has been shown to promote cell migration. Furthermore, peptide-mediated inhibition of WASF3 also resulted in the dysregulation of known downstream targets such as MMP-9 and KISS1. Finally, we demonstrate that this invasive phenotype is specific to WASF3 as depletion of WASF1 and WASF2, which can also bind to CYFIP1, did not affect invasion. Collectively, our findings suggest that targeting WASF3 function with WAHM peptides could represent a promising therapeutic strategy for preventing tumor invasion and metastasis. INTRODUCTION WASF3 (1) is one of three genes in the Wiskott-Aldridge Syndrome family which have been implicated in the regulation of cell movement related to wound healing, neuronal migration, chemotaxis and immune cell activation, through control of membrane protrusions resulting from reorganization of the actin cytoskeleton (2- 4). The protein C-termini carry motifs (VCA) that bind to monomeric actin and the ARP2/3 complex, to facilitate actin polymerization and cytoskeleton reorganization (1). In resting cells, WASF proteins are maintained in a conformation-restricted, inactive form as a result of binding the WASF regulatory complex (WRC) comprised of the CYFIP1/SRA1 (or the PIR121/CYFIP2 ortholog), NCKAP1/NAP1/HEM2 (or the HEM1 ortholog), ABI2 (or the ABI1 and ABI3 orthologs) and HSPC300/BRICK1 proteins (5-7). Activation of WASF proteins are facilitated by binding Rac proteins and phosphorylation of tyrosine residues, which leads to relaxation of the conformational constraints and, in the cases of WASF3, results in increased migration, invasion and metastasis (5). 2 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2015 American Association for Cancer Research. Author Manuscript Published OnlineFirst on December 16, 2015; DOI: 10.1158/0008-5472.CAN-15-1680 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. WASF3 involvement in invasion/metastasis has largely been studied in model cell systems, but is supported by the observation that high-level WASF3 expression is associated with high-grade primary breast (5, 8) and prostate cancers (9). Knockdown of WASF3 in breast and prostate cancer cells leads to a reduction in cell invasion in vitro and metastasis in xenograft models in vivo (5, 9). Non-metastatic cells do not express WASF3 (10), but reexpression in these cells leads to acquisition of the invasion phenotype. Although primarily considered a protein that regulates actin cytoskeleton dynamics, WASF3 has also been shown to have a regulatory function that affects expression of genes involved in metastasis such as KISS1, ZEB1 and miRNA- 200s (10-12) and its activity and expression is regulated by proteins such as JAK2, HSP70, ABL and HIF1 (13- 16), which have also been implicated in metastasis. WASF3 also interacts with the ATAD3A mitochondrial protein, which regulates its stability at the mitochondrial membrane (17). A relatively new class of inhibitors that provides the potential for much greater inhibition of protein function with high specificity has been developed, in which chemically stabilized peptides are used to target protein-protein interactions (PPIs). These “stapled peptides” (SP) are synthetically designed to stabilize and constrain an α-helical structure through macrocyclic ring formation using ring closing metathesis chemistry (18- 21). Further, these locked peptides can exhibit drug-like properties including enhanced cell permeability and resistance to proteolytic degradation (22-24). SPs have only recently been considered as biological therapeutics and still face challenges of cost and delivery but several are currently being investigated in Phase I clinical trials (25,26). The structure of the WASF proteins determines their function, which is regulated through interactions with two different subcomplexes (6,7) involving the CYFIP1-NCKAP1 dimer and the ABI2-HSPC300-WASF trimer. Regulation of the VCA domain, and hence actin polymerization, is facilitated by a complex structural interaction between CYFIP1/NCKAP1 and the WASF proteins that act allosterically to prevent actin polymerization. Analysis of the WASF1 crystal structure, and its association with the WRC proteins, demonstrates several critical interacting sites throughout the protein complex (6,7), identifying potential targeting sites to disrupt WASF3 function. In this report we describe the design of stapled peptides that target 3 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2015 American Association for Cancer Research. Author Manuscript Published OnlineFirst on December 16, 2015; DOI: 10.1158/0008-5472.CAN-15-1680 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. essential interactions between WASF3 and CYFIP1, and demonstrate that they can suppress WASF3 activation, thereby leading to loss of invasion potential in breast and prostate cancer cells without inhibiting cellular proliferation. As such, these inhibitor peptides offer an opportunity to investigate how suppression of WASF3 function can lead to suppression of invasion and metastasis. MATERIALS AND METHODS Stapled peptide synthesis Peptides were prepared manually using standard Fmoc solid-phase peptide synthesis as described previously (27). The purified peptides were quantified using the Pierce HABA-Avidin microplate protocol by measuring absorbance at 500 nm using the Biotek Synergy 2 Microplate Reader. WAHM1 molecular weight = 2291.4 (expected = 2291.8), WAHM2 molecular weight= 2305.2 (expected = 2305.8), SCR1 molecular weight = 2291.4 (expected = 2291.8), SCR2 molecular weight 2305.8 (expected = 2305.8). Molecular reagents and constructs pLKO.1 lentiviral vectors harboring shRNAs targeting WASF1, WASF2, WASF3 or NCKAP1 were obtained from Open Biosystems and shCYFIP1 was from Sigma-Aldrich. WASF2 and WASF3 antibodies were purchased from Cell Signaling Technology. Antibodies against CYFIP1, NCKAP1, WASF1, Rac1 and Rac2 were from Abcam and KISS1 was from Santa Cruz Biotechnology. Antibodies against PY20 and β-Actin were from Sigma. HSP90 inhibitor 17-AAG was obtained from Selleckchem (Houston, TX). Cell lines and standard assays MDA-MB-231 cells were obtained from ATCC (04/11) and have been verified using SNP-CGH (11) for characteristic cytogenetic changes. DU145 cells were obtained from ATCC on Feb 10, 2014 and passage <5 were used in this study. The ATCC Cell Authentication Testing service confirmed the identity of Hs578T and PC3 cells using STR DNA fingerprinting analysis (08/15/15). Lentiviral transduction, cell proliferation assays, wound healing assays, Transwell invasion assays, western blotting, flow cytometry and Real-time RT-PCR analysis were carried out as described previously (9-11,15-17). 4 Downloaded from cancerres.aacrjournals.org on September
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